Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics

Abstract

Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state.

Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein

“…Overall lower hydration in the crystalline state may lead to a lower water concentration throughout the protein scaffold including the pCa binding pocket and altered electrostatic interactions. The influence of dehydration on the later part of the photocycle of PYP has been studied in films with low hydration levels 56 and cell-mimetic environments 57 . Dehydration was found to alter both the mechanism and the kinetics of the later parts of the photocycle significantly 56,57 .…”

“…The influence of dehydration on the later part of the photocycle of PYP has been studied in films with low hydration levels 56 and cell-mimetic environments 57 . Dehydration was found to alter both the mechanism and the kinetics of the later parts of the photocycle significantly 56,57 . On earlier timescales, the isomerization quantum yield and rate have been found to be sensitive to the arrangement of water molecules inducing altered hydrogen bond interactions in the PYP active site 28 .…”

Spectrokinetic characterization of photoactive yellow protein films for integrated optical applications

“…The pG initial or dark state has an absorption maximum at 446 nm, while during the photocycle, two spectrally distinct states are present: the red-shifted pR, followed by the blue-shifted pB. Since the first study of the photocycle (Meyer et al 1987, 1989), the kinetic and spectral properties of the intermediates have been investigated with numerous methods under various environmental conditions, resulting in several distinctive photocycle schemes and rate coefficients, including spectrally “silent” transitions, too (Borucki et al 2006; Cusanovich and Meyer 2003; Groot et al 2003; Hellingwerf et al 2003; Hoff et al 1994b; Ihee et al 2005; Imamoto and Kataoka 2007; Joshi et al 2005; Kim et al 2012; Pande et al 2016; Ujj et al 1998; van der Horst et al 2005; Yang et al 2017). A recent work suggests that the protein’s photodynamics also depend on the wavelength of the excitation light (Mix et al 2018).…”

Reversible molecular motional switch based on circular photoactive protein oligomers exhibits unexpected photo-induced contraction

“…To determine the chromophore kinetics of coPYP-4, TA 49 , 50 , 83 , 84 signals at three wavelengths were measured and fitted simultaneously by using sums of four exponentials. As a result, we obtained four time constants of 1.9 ± 1.6 μs, 180 ± 60 μs, 1.4 ± 0.3 ms, and 79 ± 3 ms for coPYP-4 and 6.2 ± 6.4 μs, 280 ± 110 μs, 1.3 ± 0.6 ms, and 110 ± 3 ms for monomer PYP ( Supplemental experimental procedures ; Figure S3 ).…”